METHOD TO DETECT IgE

ABSTRACT

The present invention includes a method to detect IgE using a human Fc epsilon receptor (Fc ε R) to detect IgE antibodies in a biological sample from a cat, a dog, or a horse. The present invention also relates to kits to perform such methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. patent applicationSer. No. 11/305,447, filed Dec. 16, 2005; which is a divisional of U.S.patent application Ser. No. 10/763,400, filed Jan. 23, 2004; which is adivisional of U.S. patent application Ser. No. 09/944,277, filed Aug.30, 2001, now issued as U.S. Pat. No. 6,682,894 B2; which is adivisional of U.S. application Ser. No. 09/285,873, filed Mar. 31, 1999,now U.S. Pat. No. 6,309,832 B1; which is a divisional of U.S.application Ser. No. 08/756,387, filed Nov. 26, 1996, now issued as U.S.Pat. No. 5,945,294; each of the foregoing entitled “METHOD TO DETECTIgE” and incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to a novel method to detect epsilonimmunoglobulin (IgE). The present invention also includes novel kits todetect IgE as well as methods to produce the detection reagent.

BACKGROUND OF THE INVENTION

Diagnosis of disease and determination of treatment efficacy areimportant tools in medicine. In particular, detection of IgE productionin an animal can be indicative of disease. Such diseases include, forexample, allergy, atopic disease, hyper IgE syndrome, internal parasiteinfections and B cell neoplasia. In addition, detection of IgEproduction in an animal following a treatment is indicative of theefficacy of the treatment, such as when using treatments intended todisrupt IgE production.

Until the discovery of the present invention, detection of IgE insamples obtained from non-human animals has been hindered by the absenceof suitable reagents for detection of IgE. Various compounds have beenused to detect IgE in IgE-containing compositions. In particular,antibodies that bind selectively to epsilon idiotype antibodies (i.e.,anti-IgE antibodies) have been used to detect IgE. These anti-IgEantibodies, however, can cross-react with other antibody idiotypes, suchas gamma isotype antibodies. The discovery of the present inventionincludes the use of a Fe epsilon receptor (Fc_(ε)R) molecule to detectthe presence of IgE in a putative IgE-containing composition. A Fc_(ε)Rmolecule provides an advantage over, for example anti-IgE antibodies, todetect IgE because a Fc_(ε)R molecule can bind to an IgE with morespecificity (i.e., less idiotype cross-reactivity) and more sensitivity(i.e., affinity) than anti-IgE binding antibodies.

Lowenthal et al., 1993, Annals of Allergy 71:481-484, dog serum cantransfer cutaneous reactivity to a human. While it is possible thatLowenthal et al. properly teach the binding of human Fc_(ε)R to canineIgE. Lowenthal et al., however, do not provide data defining theparticular cellular proteins responsible for the transfer of cutaneousreactivity. As such, a skilled artisan would conclude that the transferof cutaneous reactivity taught by Lowenthal et al. could be due to avariety of different molecular interactions and that the conclusiondrawn by Lowenthal et al. is merely an interpretation. In addition,Lowenthal et al. do not teach the use of purified human Fc_(ε)R todetect canine IgE. The subunits of human Fc_(ε)R have been known asearly as 1988 and have never been used to detect canine, feline orequine IgE. Indeed, U.S. Pat. No. 4,962,035, to Leder et al., issuedOct. 9, 1990, discloses human Fc_(ε)R but does not disclose the use ofsuch a human Fc_(ε)R to detect human or non-human IgE. The use ofpurified human Fc_(ε)R avoids complications presented by use of Fc_(ε)Rbound to a cell, such as non-specific binding of the Fc_(ε)R-bearingcell due to additional molecules present on the cell membrane. Thatpurified human Fc_(ε)R detects non-human IgE is unexpected becauseinter-species binding between a Fc_(ε)R and an IgE is not predictable.For example, human Fc_(ε)R binds to rat IgE hut rat Fc_(ε)R does notbind to human IgE.

The high affinity Fc_(ε)R consists of three protein chains, alpha, betaand gamma. Prior investigators have disclosed the nucleic acid sequencefor: the alpha chain (Kochan et al., Nucleic Acids Res. 16:3584, 1988;Shimizu et al., Proc. Natl. Acad. Sci. USA 85:1907-1911, 1988; and Panget al., J. Immunol. 151:6166-6174, 1993); the beta chain (Kuster et al.,J. Biol. Chem. 267: 12782-12787, 1992); and the gamma chain (Kuster etal., J. Biol. Chem. 265-6448-6452, 1990).

Thus, methods and kits are needed in the art that will provide specificdetection of non-human IgE.

SUMMARY OF THE INVENTION

The present invention includes detection methods and kits that detectIgE. One embodiment of the present invention is a method to detect IgEcomprising: (a) contacting an isolated human Fc_(ε) receptor (Fc_(ε)R)molecule with a putative IgE-containing composition under conditionssuitable for formation of a Fc_(ε)R molecule:IgE complex, wherein theIgE is selected from the group consisting of canine IgE, feline IgE andequine IgE; and (b) determining the presence of IgE by detecting theFc_(ε)R molecule:IgE complex, the presence of the Fc_(ε)R molecule:IgEcomplex indicating the presence of IgE. A preferred Fc_(ε)R molecule inwhich a carbohydrate group of the Fc_(ε)R molecule is conjugated tobiotin.

Another embodiment of the present invention is a method to detect IgEcomprising: (a) contacting a recombinant cell with a putativeIgE-containing composition under conditions suitable for formation of arecombinant cell:IgE complex, in which the recombinant cell includes: arecombinant cell expressing a human Fc_(ε)R molecule; and a recombinantcell expressing an antibody that binds selectively to an IgE includingcanine IgE, feline IgE and equine IgE; and (b) determining the presenceof IgE by detecting the recombinant cell:IgE complex, the presence ofthe recombinant cell:IgE complex indicating the presence of IgE. Apreferred recombinant cell includes a RBL-hFc_(ε)R cell.

Another embodiment of the present invention is a method to detect fleaallergy dermatitis comprising, (a) immobilizing a flea allergen on asubstrate; (b) contacting the flea allergen with a putativeIgE-containing composition under conditions suitable for formation of anantigen:IgE complex bound to said substrate; (c) removing non-boundmaterial from the substrate under conditions that retain antigen:IgEcomplex binding to the substrate; and (c) detecting the presence of theantigen:IgE complex by contacting the antigen:IgE complex with a Fc_(ε)Rmolecule. Preferably, the flea allergen is a flea saliva antigen andmore preferably flea saliva products and/or flea saliva proteins.

The present invention also includes a kit for performing methods of thepresent invention. One embodiment is a kit for detecting IgE comprisinga human Fc_(ε) receptor (Fc_(ε)R) molecule and a means for detecting anIgE including canine IgE, feline IgE and equine IgE. Another embodimentis a general allergen kit comprising an allergen common to all regionsof the United States and a human Fc_(ε) receptor (Fc_(ε)R) molecule.Another embodiment is a kit for detecting flea allergy dermatitiscomprising a human Fc_(ε) receptor (Fc_(ε)R) molecule and a fleaallergen.

Another embodiment of the present invention is an isolated human Fc_(ε)receptor (Fc_(ε)R) alpha chain protein, in which a carbohydrate group ofthe Fc_(ε)R alpha chain protein is conjugated to biotin. A preferredFc_(ε)R alpha chain protein comprises PhFc_(ε)Rα₁₇₂-BIOT.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts ELISA results using biotinylated alpha chain of humanFc_(ε)R to detect canine IgE antibodies.

FIG. 2 depicts ELISA results using biotinylated alpha chain of humanFc_(ε)R to detect plant allergen-specific canine IgE antibodies.

FIG. 3 depicts ELISA results using biotinylated alpha chain of humanFc_(ε)R to detect human or canine IgE antibodies.

FIG. 4 depicts ELISA results using biotinylated alpha chain of humanFc_(ε)R to detect flea allergen-specific canine IgE antibodies.

FIG. 5 depicts ELISA results using biotinylated alpha chain of humanFc_(ε)R to detect flea allergen-specific and heartworm antigen-specificcanine IgE antibodies.

FIG. 6 depicts ELISA results using biotinylated alpha chain of humanFc_(ε)R to detect flea saliva-specific canine IgE antibodies.

FIG. 7 depicts ELISA results using biotinylated alpha chain of humanFc_(ε)R to detect heartworm antigen-specific feline IgE antibodies.

FIG. 8 depicts ELISA results using biotinylated alpha chain of humanFc_(ε)R to detect heartworm antigen-specific feline IgE antibodies.

FIG. 9 depicts ELISA results using biotinylated alpha chain of humanFc_(ε)R to detect antigen-specific equine IgE antibodies.

FIG. 10 depicts ELISA results using basophilic leukemia cells expressingalpha chain of human Fc_(ε)R to detect canine IgE antibodies in serafrom heartworm-infected dogs.

FIG. 11 depicts ELISA results using basophilic leukemia cells expressingalpha chain of human Fc_(ε)R to detect canine IgE antibodies in serafrom flea saliva sensitized dogs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery that purified highaffinity human Fc epsilon receptor (i.e., Fc_(ε)RI; referred to hereinas Fc_(ε)R) can be used in certain non-human (i.e., canine, feline orequine) epsilon immunoglobulin (referred to herein as IgE or IgEantibody)-based detection (e.g., diagnostic, screening) methods andkits. The use of human Fc_(ε)R to detect non-human IgE is unexpectedbecause canine, feline and equine immune systems are different from thehuman immune system, as well as from each other (i.e., moleculesimportant to the immune system usually are species specific).

One embodiment of the present invention is a method to detect anon-human IgE using an isolated human Fc_(ε)R molecule. It is to benoted that the term “a” entity or “an” entity refers to one or more ofthat entity; for example, a protein refers to one or more proteins or atleast one protein. As such, the terms “a” (or “an”), “one or more” and“at least one” can be used interchangeably herein. It is also to benoted that the terms “comprising”, “including”, and “having” can be usedinterchangeably. It is also to be noted that the terms “comprising”,“including”, and “having” can be used interchangeably. Furthermore, acompound “selected from the group consisting of” refers to one or moreof the compounds in the list that follows, including mixtures (i.e.,combinations) of two or more of the compounds.

According to the present invention, an isolated, or biologically pure,Fc_(ε)R molecule, is a molecule that has been removed from its naturalmilieu. As such, “isolated” and “biologically pure” do not necessarilyreflect the extent to which the molecule has been purified. An isolatedhuman Fc_(ε)R molecule of the present invention can be obtained from itsnatural source (e.g., from a human mast cell), can be produced usingrecombinant DNA technology or can be produced by chemical synthesis.

A Fc_(ε)R molecule (also referred to herein as Fc_(ε)R or Fc_(ε)Rprotein) of the present invention can be a full-length protein, aportion of a full-length protein or any homolog of such a protein. Asused herein, a protein can be a polypeptide or a peptide. A Fc_(ε)Rmolecule of the present invention can comprise a complete Fc_(ε)R (i.e.,alpha, beta and gamma Fc_(ε)R chains), an alpha Fc_(ε)R chain (alsoreferred to herein as Fc_(ε)R a chain) or portions thereof. Preferably,a Fc_(ε)R molecule comprises at least a portion of a Fc_(ε)R α chainthat binds to IgE, i.e., that is capable of forming an immunocomplexwith an IgE constant region. Preferably, a Fc_(ε)R molecule of thepresent invention binds to IgE with an affinity of about K_(A)≅10⁸, morepreferably with an affinity of about K_(A)≅10⁹ and even more preferablywith an affinity of about K_(A)≅10¹⁰.

An isolated Fc_(ε)R molecule of the present invention, including ahomolog, can be is identified in a straight-forward manner by theFc_(ε)R molecule's ability to form an immunocomplex with an IgE.Examples of Fc_(ε)R homologs include Fc_(ε)R proteins in which aminoacids have been deleted (e.g., a truncated version of the protein, suchas a peptide), inserted, inverted, substituted and/or derivatized (e.g.,by glycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitoylation, amidation and/or addition ofglycerophosphatidyl inositol) such that the homolog includes at leastone epitope capable of forming an immunocomplex with an IgE.

Fc_(ε)R homologs can be the result of natural allelic variation ornatural mutation. Fc_(ε)R homologs of the present invention can also beproduced using techniques known in the art including, but not limitedto, direct modifications to the protein or modifications to the geneencoding the protein using, for example, classic or recombinant DNAtechniques to effect random or targeted mutagenesis.

According to the present invention, a human Fc_(ε)R a chain of thepresent invention is encoded by at least a portion of the nucleic acidsequence of the coding strand of a cDNA encoding a full-length Fc_(ε)R αchain protein represented herein as SEQ ID NO:1, the portion at leastencoding the IgE binding site of the Fc_(ε)R a chain protein. Thedouble-stranded nucleic acid molecule including both the coding strandhaving SEQ ID NO:1 and the complementary non-coding strand (the nucleicacid sequence of which can be readily determined by one skilled in theart and is shown herein as SEQ ID NO:3) is referred to herein as Fc_(ε)Rnucleic acid molecule nhFc_(ε)Rα₁₁₉₈. Translation of SEQ ID NO:1suggests that nucleic acid molecule nhFc_(ε)Rε₁₁₉₈ encodes a full-lengthFc_(ε)R α chain protein of about 257 amino acids, referred to herein asPhFc_(ε)Rα₂₅₇, represented by SEQ ID NO:2, assuming an open readingframe having an initiation (start) codon spanning from about nucleotide107 through about nucleotide 109 of SEQ ID NO:1 and a termination (stop)codon spanning from about nucleotide 878 through about nucleotide 880 ofSEQ ID NO:1. The coding region encoding PhFc_(ε)Rα₂₅₇, including thestop codon, is represented by nucleic acid molecule nhFc_(ε)Rα₇₇₄,having a coding strand with the nucleic acid sequence represented hereinas SEQ ID NO:4. SEQ ID NO:1 encodes a signal peptide of about 25 aminoacids as well as a mature protein of about 232 amino is acids, denotedherein as PhFc_(ε)Rα₂₃₂, the amino acid sequence of which is representedherein as SEQ ID NO:6. The nucleic acid molecule encoding the apparentmature protein is referred to as nhFc_(ε)Rα₆₉₉, the nucleic acidsequence of the coding strand of which is denoted herein as SEQ E) NO:7.SEQ ID NO:1 also encodes a hydrophobic transmembrane domain and acytoplasmic tail which as a group extend from about amino acid 205 toabout amino acid 257 of SEQ ID NO:2. Knowledge of these nucleic acid andamino acid sequences allows one skilled in the art to make modificationsto the respective nucleic acid molecules and proteins to, for example,develop a Fc_(ε)R α chain protein with increased solubility and/or atruncated protein (e.g., a peptide) capable of detecting IgE, e.g.,PhFc_(ε)Rα₁₉₇ and PhFc_(ε)Rα₁₇₂. Preferred Fc_(ε)R molecules includePhFc_(ε)Rα257, PhFc_(ε)Rα₁₉₇, PhFc_(ε)Rα₂₃₂ and PhFc_(ε)Rα₁₇₂. Preferrednucleic acid molecules to encode a Fc_(ε)R molecules includenhFc_(ε)Rα₇₇₄, nhFc_(ε)Rα₁₁₉₈, nhFc_(ε)Rα₆₁₂, nhFc_(ε)Rα₅₉₁,nhFc_(ε)Rα₆₉₉ and/or nhFc_(ε)Rα₅₁₆.

Isolated Fc_(ε)R molecule protein of the present invention can beproduced by culturing a cell capable of expressing the protein underconditions effective to produce the protein, and recovering the protein.A preferred cell to culture is a recombinant cell that is capable ofexpressing the protein, the recombinant cell being produced bytransforming a host cell with one or more nucleic acid molecules of thepresent invention. Transformation of a nucleic acid molecule into a cellcan be accomplished by any method by which a nucleic acid molecule canbe inserted into the cell. Transformation techniques include, but arenot limited to, transfection, electroporation, microinjection,lipofection, adsorption, and protoplast fusion. A recombinant cell mayremain unicellular or may grow into a tissue, organ or a multicellularorganism. Transformed nucleic acid molecules of the present inventioncan remain extrachromosomal or can integrate into one or more siteswithin a chromosome of the transformed (i.e., recombinant) cell in sucha manner that their ability to be expressed is retained. Suitable andpreferred nucleic acid molecules with which to transform a cell are asdisclosed herein for suitable and preferred Fc_(ε)R nucleic acidmolecules per se. Particularly preferred nucleic acid molecules toinclude in recombinant cells of the present invention includenhFc_(ε)Rα₇₇₄, nhFc_(ε)Rα₁₁₉₈, nhFc_(ε)Rα₆₁₂, nhFc_(ε)Rα₅₉₁,nhFc_(ε)Rα₆₉₉ and/or nhFc_(ε)Rα₅₁₆.

Suitable host cells to transform include any cell that can betransformed with a nucleic acid molecule of the present invention. Hostcells can be either untransformed cells or cells that are alreadytransformed with at least one nucleic acid molecule. Host cells of thepresent invention either can be endogenously (i.e., naturally) capableof producing a Fc_(ε)R molecule protein of the present invention or canbe capable of producing such proteins after being transformed with atleast one nucleic acid molecule of the present invention. Host cells ofthe present invention can be any cell capable of producing at least oneprotein of the present invention, and include bacterial, fungal(including yeast), parasite (including protozoa and ectoparasite),insect, other animal and plant cells.

Preferably, a recombinant cell is transfected with a recombinantmolecule of the present invention is a molecule that can include atleast one of any nucleic acid molecule heretofore described operativelylinked to at least one of any transcription control sequence capable ofeffectively regulating expression of the nucleic acid molecule(s) in thecell to be transformed, examples of which are disclosed herein. Aparticularly preferred recombinant molecule includes pVL-nhFc_(ε)Rα₆₁₂.Details regarding the production of Fc_(ε)R molecule nucleic acidmolecule-containing recombinant molecules are disclosed herein.Particularly preferred recombinant cell of the present inventionincludes Trichoplusia ni-pVL-nhFc_(ε)Rα₆₁₂.

A Fc_(ε)R molecule of the present invention can include chimericmolecules comprising a portion of a Fc_(ε)R molecule that binds to anIgE and a second molecule that enables the chimeric molecule to be boundto a substrate in such a manner that the Fc_(ε)R portion binds to IgE inessentially the same manner as a Fc_(ε)R molecule that is not bound to asubstrate. An example of a suitable second molecule includes a portionof an immunoglobulin molecule.

A Fc_(ε)R molecule of the present invention can be contained in aformulation, herein referred to as a Fc_(ε)R formulation. For example, aFc_(ε)R can be combined with a buffer in which the Fc_(ε)R issolubilized, and/or a carrier. Suitable buffers and carriers are knownto those skilled in the art. Examples of suitable buffers include anybuffer in which a Fc_(ε)R can function to selectively bind to IgE, suchas, but not limited to, phosphate buffered saline, water, saline,phosphate buffer, bicarbonate buffer, HEPES buffer(N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffered saline),TES buffer (Tris-EDTA buffered saline), Tris buffer and TAE buffer(Tris-acetate-EDTA). Examples of carriers include, but are not limitedto, polymeric matrices, toxoids, and serum albumins, such as bovineserum albumin. Carriers can be in mixed with Fc_(ε)R or conjugated(i.e., attached) to Fc_(ε)R in such a manner as to not substantiallyinterfere with the ability of the Fc_(ε)R to selectively bind to IgE.

A Fc_(ε)R of the present invention can be bound to the surface of a cellexpressing the Fc_(ε)R. A preferred Fc_(ε)R-bearing cell includes arecombinant cell expressing a nucleic acid molecule encoding a humanFc_(ε)R alpha chain of the present invention. A more preferredrecombinant cell of the present invention expresses a nucleic acidmolecule that encodes at least one of the following proteins:PhFc_(ε)Rα₂₅₇ and PhFc_(ε)Rα₂₃₂. An even more preferred recombinant cellexpresses a nucleic acid molecule including nhFc_(ε)Rα₆₁₂,nhFc_(ε)Rα₅₉₁, nhFc_(ε)Rα₆₉₉ and/or nhFc_(ε)Rα₅₁₆ with a recombinantcell expressing a nucleic acid molecule comprising a nucleic acidsequence including SEQ ID NO:1 or SEQ ID NO:4, or a nucleic acidmolecule comprising an allelic variant of a nucleic acid moleculecomprising SEQ ID NO:1 or SEQ ID NO:4, being even more preferred. Aneven more preferred recombinant cell is a RBL-hFc_(ε)R cell.

In addition, a Fc_(ε)R formulation of the present invention can includenot only a Fc_(ε)R but also one or more additional antigens orantibodies useful in detecting IgE. As used herein, an antigen refers toany molecule capable of being selectively bound by an antibody. As usedherein, specific binding of a first molecule to a second molecule torefers to the ability of the first molecule to preferentially bind(e.g., having higher affinity higher avidity) to the second moleculewhen compared to the ability of a first molecule to bind to a thirdmolecule. The first molecule need not necessarily be the natural ligandof the second molecule. Examples of such antibodies include, but are notlimited to, antibodies that bind selectively to the constant region ofan IgE heavy (i.e., anti-IgE isotype antibody) or antibodies that bindselectively to an IgE having a specific antigen specificity (i.e.,anti-IgE idiotypic antibody). Examples of such antigens include anyantigen known to induce the production of IgE. Preferred antigensinclude allergens and parasite antigens. Allergens of the presentinvention are preferably derived from fungi, trees, weeds, shrubs,grasses, wheat, corn, soybeans, rice, eggs, milk, cheese, bovines (orcattle), poultry, swine, sheep, yeast, fleas, flies, mosquitos, mites,midges, biting gnats, lice, bees, wasps, ants, true bugs or ticks. Asuitable flea allergen includes an allergen derived from a flea, inparticular flea saliva antigen. A preferred flea allergen includes aflea saliva antigen Preferred flea saliva antigens include antigens suchas those disclosed in PCT Patent Publication No. WO 96/11271, publishedApr. 18, 1996, by Frank et al. (this publication is incorporated byreference herein in its entirety), with flea saliva products and fleasaliva proteins being particularly preferred. According to the presentinvention, a flea saliva protein includes a protein produced byrecombinant DNA methods, as well as proteins isolated by other methodsdisclosed in PCT Patent Publication No. WO 96/11271.

Preferred general allergens include those derived from grass, MeadowFescue, Curly Dock, plantain, Mexican Firebush, Lamb's Quarters,pigweed, ragweed, sage, elm, cocklebur, Box Elder, walnut, cottonwood,ash, birch, cedar, oak, mulberry, cockroach, Dermataphagoides,Atternaria, Aspergillus, Cladosporium, Fusarium, Helminthosporium,Mucor, Penicillium, Pulu/alria, Rhizopus and/or Tricophyton. Morepreferred general allergens include those derived from Johnson Grass,Kentucky Blue Grass, Meadow Fescue, Orchard Grass, Perennial Rye Grass,Redtop Grass, Timothy Grass, Bermuda Grass, Brome Grass, Curly Dock,English Plantain, Mexican Firebush, Lamb's Quarters, Rough Pigweed ShortRagweed, Wormwood Sage, American Elm, Common Cocklebur, Box Elder, BlackWalnut, Eastern Cottonwood, Green Ash, River Birch, Red Cedar, Red Oak,Red Mulberry, Cockroach, Dermataphagoides farinae, Alternaria alternata,Aspergillus ummigatus, Cladosporium herbarum, Fusarium vasinfectum,Helminthosporium sativum, Mucor recemosus, Penicillium notatum,Pulll/aria pullulans, Rhizopus nigricans and/or Tricophyton spp.Preferred parasite antigens include, but are not limited to, helminthantigens, in particular heartworm antigens, such as Di33 (described inU.S. patent application Ser. No. 08/715,628, filed Sep. 18, 1996, toGrieve et al.). The term “derived from” refers to a natural allergen ofsuch plants or organisms (i.e., an allergen directly isolated from suchplants or organisms), as well as, non-natural allergens of such plantsor organisms that posses at least one epitope capable of eliciting animmune response against an allergen (e.g., produced using recombinantDNA technology or by chemical synthesis).

The present invention also includes human Fc_(ε)R mimetopes and usethereof to detect IgE. In accordance with the present invention, a“mimetope” refers to any compound that is able to mimic the ability of aFc_(ε)R molecule to bind to IgE. A mimetope can be a peptide that hasbeen modified to decrease its susceptibility to degradation but thatstill retains IgE-binding activity. Other examples of mimetopes include,but are not limited to, carbohydrate-based compounds, lipid-basedcompounds, nucleic acid-based compounds, natural organic compounds,synthetically derived organic compounds, anti-idiotypic antibodiesand/or catalytic antibodies, or fragments thereof. A mimetope can beobtained by, for example, screening libraries of synthetic compounds forcompounds capable of binding to IgE. A mimetope can also be obtained by,for example, rational drug design. In a rational drug design procedure,the three-dimensional structure of a compound of the present inventioncan be analyzed by, for example, nuclear magnetic resonance (NMR) orx-ray crystallography. The three-dimensional structure can then be usedto predict structures of potential mimetopes by, for example, computermodeling. The predicted mimetope structures can then be produced by, forexample, chemical synthesis, recombinant DNA technology, or by isolatinga mimetope from a natural source. Specific examples of Fc_(ε)R mimetopesinclude anti-idiotypic antibodies, oligonucleotides produced using Selextechnology, peptides identified by random screening of peptide librariesand proteins identified by phage display technology.

One embodiment of the present invention is a method to detect non-humanIgE which includes the steps of: (a) contacting an isolated human Fc_(ε)receptor (Fc_(ε)R) molecule with a putative IgE-containing compositionunder conditions suitable for formation of an Fc_(ε)R molecule:IgEcomplex; and (b) detecting levels of IgE by detecting said Fc_(ε)Rmolecule:IgE complex. Presence of such a Fc_(ε)R molecule:IgE complexindicates that the animal is producing IgE. Preferred non-human IgE todetect using a human Fc_(ε)R molecule include canine IgE, feline IgE andequine IgE. The present method can further include the step ofdetermining whether an IgE complexed with a Fc_(ε)R molecule is heatlabile. Methods to determine heat lability of IgE are disclosed in theExamples section. Preferably, an IgE is heat labile when incubated atabout 56° C. for about 4 hours. Without being bound by theory,Applicants believe that heat labile forms of IgE bind to certainallergens and non-heat labile forms of IgE bind to other types ofallergens. As such, detection of heat labile IgE compared with non-heatlabile IgE can be used to discriminate between allergen sensitivities.For example, Applicants believe that IgE antibodies that bind to certainflea allergens and heartworm allergens are heat labile while IgEantibodies that bind to certain plant allergens are not heat labile.Thus, the presence of non-heat labile IgE can indicate that an animal issensitive to certain plant allergens but not to certain flea orheartworm allergens. Moreover, Applicants believe that identification ofheat labile IgE and non-heat labile IgE using a human Fc_(ε)R suggeststhe presence of different sub-populations of IgE that may or may nothave substantially similar structures to known IgE. As such, a Fc_(ε)Rmolecule of the present invention may be useful for detecting moleculesbound by the Fc_(ε)R molecule but not identical to a known IgE.

As used herein, canine refers to any member of the dog family, includingdomestic dogs, wild dogs and zoo dogs. Examples of dogs include, but arenot limited to, domestic dogs, wild dogs, foxes, wolves, jackals andcoyotes. As used herein, a feline refers to any member of the catfamily, including domestic cats, wild cats and zoo cats. Examples ofcats include, but are not limited to, domestic cats, lions, tigers,leopards, panthers, cougars, bobcats, lynx, jaguars, cheetahs, andservals. As used herein, equine refers to any member of the horsefamily, including horses, donkeys, mules and zebras.

As used herein, the term “contacting” refers to combining or mixing, inthis case a putative IgE-containing composition with a human Fc_(ε)Rmolecule. Formation of a complex between a Fc_(ε)R and an IgE refers tothe ability of the Fc_(ε)R to selectively bind to the IgE in order toform a stable complex that can be measured (i.e., detected). As usedherein, the term selectively binds to an IgE refers to the ability of aFc_(ε)R of the present invention to preferentially bind to IgE, withoutbeing able to substantially bind to other antibody isotypes. Bindingbetween a Fc_(ε)R and an IgE is effected under conditions suitable toform a complex; such conditions (e.g., appropriate concentrations,buffers, temperatures, reaction times) as well as methods to optimizesuch conditions are known to those skilled in the art, and examples aredisclosed herein. Examples of complex formation conditions are alsodisclosed in, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Labs Press, 1989, the referenceSambrook et al., ibid., is incorporated by reference herein in itsentirety.

As used herein, the term “detecting complex formation” refers todetermining if any complex is formed, i.e., assaying for the presence(i.e., existence) of a complex. If complexes are formed, the amount ofcomplexes formed can, but need not be, determined. Complex formation, orselective binding, between Fc_(ε)R and any IgE in the composition can bemeasured (i.e., detected, determined) using a variety of methodsstandard in the art (see, for example, Sambrook et al. ibid.), examplesof which are disclosed herein.

In one embodiment, a putative IgE-containing composition of the presentmethod includes a biological sample from an animal. A suitablebiological sample includes, but is not limited to, a bodily fluidcomposition or a cellular composition. A bodily fluid refers to anyfluid that can be collected (i.e., obtained) from an animal, examples ofwhich include, but are not limited to, blood, serum, plasma, urine,tears, aqueous humor, central nervous system fluid (CNF), saliva, lymph,nasal secretions, milk and feces. Such a composition of the presentmethod can, but need not be, pretreated to remove at least some of thenon-IgE isotypes of immunoglobulin and/or other proteins, such asalbumin, present in the fluid. Such removal can include, but is notlimited to, contacting the bodily fluid with a material, such as ProteinG, to remove IgG antibodies and/or affinity purifying IgE antibodiesfrom other components of the body fluid by exposing the fluid to, forexample, Concanavalin A. In another embodiment, a composition includescollected bodily fluid that is pretreated to concentrate immunoglobulincontained in the fluid. For example, immunoglobulin contained in abodily fluid can be precipitated from other proteins using ammoniumsulfate. A preferred composition of the present method is serum.

In another embodiment, a composition of the present method includes anIgB-producing cell. Such a cell can have IgE bound to the surface of thecell and/or can secrete IgE. Examples of such cells include basophilcells and myeloma cells. IgE can be bound to the surface of a celleither directly to the membrane of a cells or bound to a molecule (e.g.,an antigen) bound to the surface of the cell.

A complex can be detected in a variety of ways including, but notlimited to use of one or more of the following assays: an enzyme-linkedimmunoassay, a radioimmunoassay, a fluorescence immunoassay, achemiluminescent assay, a lateral flow assay, an agglutination assay, aparticulate-based assay (e.g., using particulates such as, but notlimited to, magnetic particles or plastic polymers, such as latex orpolystyrene beads), an immunoprecipitation assay, a BioCore™ assay(e.g., using colloidal gold) and an immunoblotting assay (e.g., awestern blot). Such assays are well known to those skilled in the art.Assays can be used to give qualitative or quantitative results dependingon how they are used. Some assays, such as agglutination, particulateseparation, and immunoprecipitation, can be observed visually (e.g.,either by eye or by a machines, such as a densitometer orspectrophotometer) without the need for a detectable marker. In otherassays, conjugation (i.e., attachment) of a detectable marker to theFc_(ε)R or to a reagent that selectively binds to the Fc_(ε)R or to theIgE being detected (described in more detail below) aids in detectingcomplex formation. Examples of detectable markers include, but are notlimited to, a radioactive label, a fluorescent label, a chemiluminescentlabel, a chromophoric label or a ligand. A ligand refers to a moleculethat binds selectively to another molecule. Preferred detectable markersinclude, but are not limited to, fluorescein, a radioisotope, aphosphatase (e.g., alkaline phosphatase), biotin, avidin, a peroxidase(e.g., horseradish peroxidase) and biotin-related compounds oravidin-related compounds (e.g., streptavidin or ImmunoPure®NeutrAvidin). Preferably, biotin is conjugated to an alpha chain of aFc_(ε)R. Preferably a carbohydrate group of the Fc_(ε)R alpha chain isconjugated to biotin. A preferred Fc_(ε)R molecule conjugated to biotincomprises PhFc_(ε)Rα₁₇₂-BIOT (the production of which is described inthe Examples section).

In one embodiment, a complex is detected by contacting a putativeIgE-containing composition with a Fc_(ε)R molecule that is conjugated toa detectable marker. A suitable detectable marker to conjugate to aFc_(ε)R molecule includes, but is not limited to, a radioactive label, afluorescent label, a chemiluminescent label or a chromophoric label. Adetectable marker is conjugated to a Fc_(ε)R molecule or a reagent insuch a manner as not to block the ability of the Fc_(ε)R or reagent tobind to the IgE being detected. Preferably, a carbohydrate group of aFc_(ε)R is conjugated to biotin.

In another embodiment, a Fc_(ε)R molecule:IgE complex is detected bycontacting a putative IgE-containing composition with a Fc_(ε)R moleculeand then contacting the complex with an indicator molecule. Suitableindicator molecules of the present invention include molecules that canbind to either the Fc_(ε)R molecule or to the IgE antibody. As such, anindicator molecule can comprise, for example, a Fc_(ε)R molecule, anantigen, an antibody and a lectin, depending upon which portion of theFc_(ε)R molecule:IgE complex being detected. Preferred identifyinglabeled compounds that are antibodies include, for example, anti-IgEantibodies and anti-Fc_(ε)R antibodies. Preferred lectins include thoselectins that bind to high-mannose groups. More preferred lectins bind tohigh-mannose groups present on a Fc_(ε)R molecule of the presentinvention produced in insect cells. An indicator molecule itself can beattached to a detectable marker of the present invention. For example,an antibody can be conjugated to biotin, horseradish peroxidase,alkaline phosphatase or fluorescein.

In one preferred embodiment, a Fc_(ε)R molecule:IgE complex is detectedby contacting the complex with a reagent that selectively binds to aFc_(ε)R molecule of the present invention. Examples of such a reagentincludes, but are not limited to, an antibody that selectively binds toa Fc_(ε)R molecule (referred to herein as an anti-Fc_(ε)R antibody) or acompound that selectively binds to a detectable marker conjugated to aFc_(ε)R molecule. Fc_(ε)R molecules conjugated to biotin are preferablydetected using streptavidin, more preferably using ImmunoPure®NeutrAvidin (available from Pierce, Rockford, Ill.).

In another preferred embodiment, a Fc_(ε)R molecule:IgE complex isdetected by contacting the complex with a reagent that selectively bindsto an IgE antibody (referred to herein as an anti-IgE reagent). Examplesof such an anti-IgE reagent include, but are not limited to, a secondaryantibody that is an anti-isotype antibody (e.g., an antibody thatselectively binds to the constant region of an IgE), an antibody-bindingbacterial surface protein (e.g., Protein A or Protein G), anantibody-binding cell (e.g., a B cell, a T cell, a natural killer cell,a polymorphonuclear leukocyte cell, a monocyte cell or a macrophagecell), an antibody-binding eukaryotic cell surface protein (e.g., an Fcreceptor), and an antibody-binding complement protein. Preferredanti-IgE reagents include, but are not limited to, D9, and CMI antibody#9, CMI antibody #19, CMI antibody #59 and CMI antibody #71 (availablefrom Custom Monoclonal International, West Sacramento, Calif.). Inparticular, as used herein, an anti-IgE antibody includes not only acomplete antibody but also any subunit or portion thereof that iscapable of selectively binding to an IgE heavy chain constant region.For example, a portion of an anti-IgE reagent can include an Fabfragment or a F(ab′)₂ fragment, which are described in detail in Janewayet al., in Immunobiology, the Immune System in Health and Disease,Garland Publishing, Inc., NY, 1996 (which is incorporated herein by thisreference in its entirety).

In one embodiment a complex can be formed and detected in solution. Inanother embodiment, a complex can be formed in which one or more membersof the complex are immobilized on (e.g., coated onto) a substrate.Immobilization techniques are known to those skilled in the art.Suitable substrate materials include, but are not limited to, plastic,glass, gel, celluloid, paper, PVDF (poly-vinylidene-fluoride), nylon,nitrocellulose, and particulate materials such as latex, polystyrene,nylon, nitrocellulose, agarose and magnetic resin. Suitable shapes forsubstrate material include, but are not limited to, a well (e.g.,microtiter dish well), a plate, a dipstick, a bead, a lateral flowapparatus, a membrane, a filter, a tube, a dish, a celluloid-typematrix, a magnetic particle, and other particulates, A particularlypreferred substrate comprises an ELISA plate, a dipstick, aradioimmunoassay plate, agarose beads, plastic beads, latex beads,immunoblot membranes and immunoblot papers. In one embodiment, asubstrate, such as a particulate, can include a detectable marker.

A preferred method to detect IgE is an immunosorbent assay. Animmunoabsorbent assay of the present invention comprises a capturemolecule and an indicator molecule. A capture molecule of the presentinvention binds to an IgE in such a manner that the IgE is immobilizedto a substrate. As such, a capture molecule is preferably immobilized toa substrate of the present invention prior to exposure of the capturemolecule to a putative IgE-containing composition. An indicator moleculeof the present invention detects the presence of an IgE bound to acapture molecule. As such, an indicator molecule preferably is notimmobilized to the same substrate as a capture molecule prior toexposure of the capture molecule to a putative IgE-containingcomposition.

A preferred immunoabsorbent assay method includes a step of either: (a)binding an Fc_(ε)R molecule to a substrate prior to contacting a Fc_(ε)Rmolecule with a putative IgE-containing composition to form a Fc_(ε)Rmolecule-coated substrate; or (b) binding a putative IgE-containingcomposition to a substrate prior to contacting a Fc_(ε)R molecule with aputative IgE-containing composition to form a putative IgE-containingcomposition-coated substrate. Preferably, the substrate includes of anon-coated substrate, a Fc_(ε)R molecule-coated substrate, anantigen-coated substrate or an anti-IgE antibody-coated substrate.

Both a capture molecule and an indicator molecule of the presentinvention are capable of binding to an IgE. Preferably, a capturemolecule binds to a different region of an IgE than an indicatormolecule, thereby allowing a capture molecule to be bound to an IgE atthe same time as an indicator molecule. The use of a reagent as acapture molecule or an indicator molecule depends upon whether themolecule is immobilized to a substrate when the molecule is exposed toan IgE. For example, a Fc_(ε)R molecule of the present invention is usedas a capture molecule when the Fc_(ε)R molecule is bound to a substrate.Alternatively, a Fc_(ε)R molecule is used as an indicator molecule whenthe Fc_(ε)R molecule is not bound to a substrate. Suitable molecule foruse as capture molecules or indicator molecules include, but are notlimited to, a Fc_(ε)R molecule of the present invention, an antigenreagent or an anti-IgE antibody reagent of the present invention.

An immunoabsorbent assay of the present invention can further compriseone or more layers and/or types of secondary molecules or other bindingmolecules capable of detecting the presence of an indicator molecule.For example, an untagged (i.e., not conjugated to a detectable marker)secondary antibody that selectively binds to an indicator molecule canbe bound to a tagged (i.e., conjugated to a detectable marker) tertiaryantibody that selectively binds to the secondary antibody. Suitablesecondary antibodies, tertiary antibodies and other secondary ortertiary molecules can be selected by those of skill in the art.Preferred secondary molecules of the present invention include, anantigen, an anti-IgE idiotypic antibody and an anti-IgE isotypic.Preferred tertiary molecules can be selected by a skilled artisan basedupon the characteristics of the secondary molecule. The same strategy isapplied for subsequent layers.

In one embodiment, a desired antigen is used as a capture molecule bybeing immobilized on a substrate, such as a microtiter dish well or adipstick. Preferred antigens include those disclosed herein. Abiological sample collected from an animal is applied to the substrateand incubated under conditions suitable (i.e., sufficient) to allow forantigen:IgE complex formation bound to the substrate (i.e., IgE in asample binds to an antigen immobilized on a substrate). Excess non-boundmaterial (i.e., material from the biological sample that has not boundto the antigen), if any, is removed from the substrate under conditionsthat retain antigen:IgE complex binding to the substrate. Preferredconditions are disclosed herein in the Examples section and generally inSambrook et al., ibid. An indicator molecule that can selectively bindto an IgE bound to the antigen, the indicator molecule can be conjugatedto a detectable marker (preferably to an enzyme label, to a colorimetriclabel, to a fluorescent label, to a radioisotope, or to a ligand such asof the biotin or avidin family), is added to the substrate and incubatedto allow formation of a complex between the indicator molecule and theantigen:IgE complex. Excess indicator molecule is removed, a developingagent is added if required, and the substrate is submitted to adetection device for analysis. A preferred indicator molecule for thisembodiment is a Fc_(ε)R molecule, preferably conjugated to biotin, to afluorescent label or to an enzyme label.

In one embodiment, a Fc_(ε)R molecule is used as a capture molecule bybeing immobilized on a substrate, such as a microtiter dish well or adipstick. A biological sample collected from an animal is applied to thesubstrate and incubated under conditions suitable to allow for Fc_(ε)Rmolecule:IgE complex formation bound to the substrate. Excess non-boundmaterial, if any, is removed from the substrate under conditions thatretain Fc_(ε)R molecule:IgE complex binding to the substrate. Anindicator molecule that can selectively bind to an IgE bound to theFc_(ε)R is added to the substrate and incubated to allow formation of acomplex between the indicator molecule and the Fc_(ε)R molecule:IgEcomplex. Preferably, the indicator molecule is conjugated to adetectable marker (preferably to an enzyme label, to a colorimetriclabel, to a fluorescent label, to a radioisotope, or to a ligand such asof the biotin or avidin family). Excess indicator molecule is removed, adeveloping agent is added if required, and the substrate is submitted toa detection device for analysis. A preferred indicator molecule for thisembodiment is an antigen that will bind to IgE in the biological sampleor an anti-IgE isotype or idiotype antibody, either preferably beingconjugated to fluorescein or biotin.

In one embodiment, an anti-IgE antibody (e.g., isotype or idiotypespecific antibody) is used as a capture molecule by being immobilized ona substrate, such as a microtiter dish well or a dipstick. A biologicalsample collected from an animal is applied to the substrate andincubated under conditions suitable to allow for anti-IgE antibody:IgEcomplex formation bound to the substrate. Excess non-bound material, ifany, is removed from the substrate under conditions that retain anti-IgEantibody:IgE complex binding to the substrate. A Fc_(ε)R molecule isadded to the substrate and incubated to allow formation of a complexbetween the Fc_(ε)R molecule and the anti-IgE antibody.IgE complex.Preferably, the Fc_(ε)R molecule is conjugated to a detectable marker(preferably to biotin, an enzyme label or a fluorescent label). ExcessFc_(ε)R molecule is removed, a developing agent is added if required,and the substrate is submitted to a detection device for analysis.

In one embodiment, an immunosorbent assay of the present invention doesnot utilize a capture molecule. In this embodiment, a biological samplecollected from an animal is applied to a substrate, such as a microtiterdish well or a dipstick, and incubated under conditions suitable toallow for IgE binding to the substrate. Any IgE present in the bodilyfluid is immobilized on the substrate. Excess non-bound material, ifany, is removed from the substrate under conditions that retain IgEbinding to the substrate. A Fc_(ε)R molecule is added to the substrateand incubated to allow formation of a complex between the Fc_(ε)Rmolecule and the IgE. Preferably, the Fc_(ε)R molecule is conjugated toa detectable marker (preferably to biotin, an enzyme label or afluorescent label). Excess Fc_(ε)R molecule is removed, a developingagent is added if required, and the substrate is submitted to adetection device for analysis.

Another preferred method to detect IgE is a lateral flow assay, examplesof which are disclosed in U.S. Pat. No. 5,424,193, issued Jun. 13, 1995,by Pronovost et al.; U.S. Pat. No. 5,415,994, issued May 16, 1995, byImrich et al; WO 94/29696, published Dec. 22, 1994, by Miller et al.;and WO 94/01775, published Jan. 20, 1994, by Pawlak et al.; each ofthese patent publications is incorporated by reference herein in itsentirety. In one embodiment, a biological sample is placed in a lateralflow apparatus that includes the following components: (a) a supportstructure defining a flow path; (b) a labeling reagent comprising a beadconjugated to an antigen, the labeling reagent being impregnated withinthe support structure in a labeling zone; and (c) a capture reagentcomprising an IgE-binding composition. Preferred antigens include thosedisclosed herein. The capture reagent is located downstream of thelabeling reagent within a capture zone fluidly connected to the labelingzone in such a manner that the labeling reagent can flow from thelabeling zone into the capture zone. The support structure comprises amaterial that does not impede the flow of the beads from the labelingzone to the capture zone. Suitable materials for use as a supportstructure include ionic (i.e., anionic or cationic) material. Examplesof such a material include, but are not limited to, nitrocellulose (NC),PVDF, carboxymethylcellulose (CM). The support structure defines a flowpath that is lateral and is divided into zones, namely a labeling zoneand a capture zone. The apparatus can further comprise a samplereceiving zone located along the flow path, more preferably upstream ofthe labeling reagent. The flow path in the support structure is createdby contacting a portion of the support structure downstream of thecapture zone, preferably at the end of the flow path, to an absorbentcapable of absorbing excess liquid from the labeling and capture zones.

In this embodiment, the biological sample is applied to the samplereceiving zone which includes a portion of the support structure. Thelabeling zone receives the sample from the sample receiving zone whichis directed downstream by the flow path. The labeling zone comprises thelabeling reagent that binds to IgE. A preferred labeling reagent is anantigen conjugated, either directly or through a linker, to a plasticbead substrate, such as to a latex bead. The substrate also includes adetectable marker, preferably a colorimetric marker. Typically, thelabeling reagent is impregnated to the support structure by drying orlyophilization. The sample structure also comprises a capture zonedownstream of the labeling zone. The capture zone receives labelingreagent from the labeling zone which is directed downstream by the flowpath. The capture zone contains the capture reagent, in this case aFc_(ε)R molecule, as disclosed above, that immobilizes the IgE complexedto the antigen in the capture zone. The capture reagent is preferablyfixed to the support structure by drying or lyophilizing. The labelingreagent accumulates in the capture zone and the accumulation is assessedvisually or by an optical detection device.

In another embodiment, a lateral flow apparatus used to detect IgEincludes: (a) a support structure defining a flow path; (b) a labelingreagent comprising a Fc_(ε)R molecule as described above, the labelingreagent impregnated within the support structure in a labeling zone; and(c) a capture reagent comprising an antigen, the capture reagent beinglocated downstream of the labeling reagent within a capture zone fluidlyconnected to the labeling zone in such a manner that the labelingreagent can flow from the labeling zone into the capture zone. Theapparatus preferably also includes a sample receiving zone located alongthe flow path, preferably upstream of the labeling reagent. Theapparatus preferably also includes an absorbent located at the end ofthe flow path.

One embodiment of the present invention is an inhibition assay in whichthe presence of IgE in a putative IgE-containing composition isdetermined by adding such composition to a Fc_(ε)R molecule of thepresent invention and an isolated IgE known to bind to the Fc_(ε)Rmolecule. The absence of binding of the Fc_(ε)R molecule to the knownIgE indicating the presence of IgE in the putative IgE-containingcomposition.

The present invention also includes kits to detect IgE based on each ofthe disclosed detection methods. One embodiment is a kit to detect IgEcomprising a human Fc_(ε) receptor (Fc_(ε)R) molecule and a means fordetecting an IgE including canine IgE, feline IgE and/or equine IgE.Suitable and preferred Fc_(ε)R molecules are disclosed herein. Suitablemeans of detection include compounds disclosed herein that bind toeither the Fc_(ε)R molecule or to an IgE. A preferred kit of the presentinvention further comprises a detection means including one or moreantigens disclosed herein, an antibody capable of selectively binding toan IgE disclosed herein and/or a compound capable of binding to adetectable marker conjugated to a Fc_(ε)R molecule (e.g., avidin,streptavidin and ImmunoPure® NeutrAvidin when the detectable marker isbiotin). Such antigens preferably induce IgE antibody production inanimals including canines, felines and/or equines.

A preferred embodiment of a kit of the present invention is a fleaallergen kit comprising a flea allergen such as those disclosed herein.A particularly preferred flea allergen for use with a flea allergen kitincludes a flea saliva product or a flea saliva protein.

Another preferred kit of the present invention is a general allergen kitcomprising an allergen common to all regions of the United States and ahuman Fc_(ε)R molecule of the present invention. As used herein, a“general allergen” kit refers to a kit comprising allergens that arefound substantially throughout the United States (i.e., essentially notlimited to certain regions of the United States). A general allergen kitprovides an advantage over regional allergen kits because a single kitcan be used to test an animal located in most geographical locations onthe United States. Suitable and preferred general allergens for use witha general allergen kit of the present invention include those generalallergens disclosed herein.

Another preferred kit of the present invention is a food allergen kitcomprising a food allergen including beef, chicken, pork, a mixture offish, such as cod, halibut or and tuna, egg, milk, Brewer's yeast, wholewheat, corn, soybean, cheese and rice, and a human Fc_(ε)R molecule ofthe present invention. Preferably, the beef, chicken, pork, fish, cornand rice, are cooked.

A preferred kit of the present invention includes those in which theallergen is immobilized to a substrate. If a kit comprises two or moreantigens, the kit can comprise one or more compositions, eachcomposition comprising one antigen. As such, each antigen can be testedseparately. A kit can also contain two or more diagnostic reagents forIgE, additional isolated IgE antigens and/or antibodies as disclosedherein. Particularly preferred are kits used in a lateral flow assayformat. It is within the scope of the present invention that a lateralflow assay kit can include one or more lateral flow assay apparatuses.Multiple lateral flow apparatuses can be attached to each other at oneend of each apparatus, thereby creating a fan-like structure.

In particular, a method and kit of the present invention are useful fordiagnosing abnormal conditions in animals that are associated withchanging levels of IgE. Particularly preferred conditions to diagnoseinclude allergies, parasitic infections and neoplasia. For example, amethod and kit of the present invention are particularly useful fordetecting flea allergy dermatitis (FAD), when such method or kitincludes the use of a flea saliva antigen. FAD is defined as ahypersensitive response to fleabites. Preferably, a putativeIgE-containing composition is obtained from an animal suspected ofhaving FAD. Preferred animals include those disclosed herein, with dogsand cats being more preferred. In addition, methods and kits of thepresent invention are particularly useful for detecting helminthinfection, in particular heartworm infection, when such methods or kitsinclude the use of a helminth antigen, such as Di33. Preferably, aputative IgE-containing composition is obtained from an animal suspectedof having a helminth infection. Preferred animals include thosedisclosed herein, with dogs and cats being more preferred.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention.

EXAMPLES Example 1

This example describes the construction of a recombinant baculovirusexpressing a truncated portion of the α-chain of the human Fc_(ε)receptor.

Recombinant molecule pVL-nhFc_(ε)Rα₆₁₂, containing a nucleic acidmolecule encoding the extracellular domain of the Fc_(ε)Rα chain,operatively linked to baculovirus polyhedron transcription controlsequences was produced in the following manner. A cDNA clone encodingthe full-length alpha chain (α chain) of the human Fc_(ε) receptor wasobtained from Dr. Jean-Pierre Kinet (Harvard University, Cambridge,Mass.). The cDNA clone included an about 1198 nucleotide insert,referred to herein as nhFc_(ε)Rα₁₁₉₈. The nucleic acid sequence of thecoding strand of nhFc_(ε)Rα₁₁₉₈ is denoted herein as SEQ ID NO:1.Translation of SEQ ID NO:1 indicates that nucleic acid moleculenhFc_(ε)Rα₁₁₉₈ encodes a full-length human Fc_(ε) receptor a chainprotein of about 257 amino acids, referred to herein as PhFc_(ε)Rα₂₅₇,having amino acid sequence SEQ ID NO:2, assuming an open reading framein which the initiation codon spans from about nucleotide 107 throughabout nucleotide 109 of SEQ ID NO:1 and the termination codon spans fromabout nucleotide 878 through about nucleotide 880 of SEQ ID NO:1. Thecomplement of SEQ ID NO:1 is represented herein by SEQ ID NO:3. Theproposed mature protein In (i.e., Fc_(ε)Rα chain from which the signalsequence has been cleaved), denoted herein as PhFc_(ε)Rα₂₃₂, containsabout 232 amino acids which is represented herein as SEQ ID NO:6. Thenucleic acid molecule encoding PhFc_(ε)Rα₂₃₂ is denoted herein asnhFc_(ε)Rα₆₉₆, the coding strand of which is represented by SEQ ID NO:7.

To produce a secreted form of the extracellular domain of the Fc_(ε)R αchain, the hydrophobic transmembrane domain and the cytoplasmic tail ofthe Fc_(ε)R α chain encoded by nhFc_(ε)Rα₁₁₉₈ were removed as follows. AFc_(ε)R α chain extracellular domain nucleic acid molecule-containingfragment of about 612 nucleotides was PCR amplified from nhFc_(ε)Rα₁₁₉₈using a forward primer EJH 040 containing a BamHI site, having thenucleic acid sequence 5′ CGC GOA TCC TAT AAT ATG GCT CCT GCC ATG G 3′(denoted SEQ ID NO:8) and a reverse primer IgE ANTI-SENSE containing anEcoRI site, having the nucleic acid sequence 5′ GGC GAA TTC TTA AGC TTTTAT TAC AG 3′ (denoted herein as SEQ ID NO:9). The resulting PCR productwas digested with BamHI and EcoRI to produce nhFc_(≢)Rα₆₁₂. Nucleic acidmolecule nhFc_(ε)Rα₆₁₂ contained an about 591 nucleotide fragmentencoding the extraceliular domain of the human Fc_(ε)R α chain,extending from about nucleotide 107 to about nucleotide 697 of SEQ ID NO1, denoted herein as nucleic acid molecule nhFc_(ε)Rα₅₉₁, the codingstrand of which has a nucleic acid sequence denoted SEQ ID NO:10.Translation of SEQ ID NO:10 indicates that nucleic acid moleculenhFc_(ε)Rα₆₁₂ encodes a Fc_(ε)R protein of about 197 amino acids,referred to herein as PhFc_(ε)Rα₁₉₇, having amino acid sequence SEQ IDNO:11. Nucleic acid molecule nhFc_(ε)Rα₆₁₂ encodes a secretable form ofthe human Fc_(ε)R α chain which does not possess a leader sequence,which is denoted herein as PhFc_(ε)Rα₁₇₂ having amino acid sequence SEQID NO:13. The coding region for PhFc_(ε)Rα₁₇₂ is denoted nhFc_(ε)Rα₅₁₆,the coding strand of which has a nucleic acid sequence denoted SEQ IDNO:12. The complement of SEQ ID NO:12 is represented herein by SEQ IDNO:14.

In order to produce a baculovirus recombinant molecule capable ofdirecting the production of PhFc_(ε)Rα₁₉₇, the nucleic acid moleculenhFc_(ε)Rα₆₁₂ was subcloned into unique BamHI and EcoRI sites of pVL1392baculovirus shuttle plasmid (available from Pharmingen, San Diego,Calif.) to produce a recombinant molecule referred to herein aspVL-nhFc_(ε)Rα₆₁₂. The resultant recombinant molecule pVL-nhFc_(ε)Rα₆₁₂was verified for proper insert orientation by restriction mapping.

Example 2

This example describes the production of PhFc_(ε)Rα₁₇₂ protein.

The recombinant molecule pVL-nhFc_(ε)Rα₆₁₂ was co-transfected with alinear Baculogold baculovirus DNA (available from Pharmingen) intoTrichoplusia ni cells (available from Invitrogen Corp., San Diego,Calif.; High Five™ cells) using the following method. About 1.5 litercultures of serum-free ex-Cell Medium (available from Invitrogen) wereseeded with about 1×10⁶ cells per ml of medium. The Trichoplusia nicells were infected with recombinant molecule pVL-nhFc_(ε)Rα₆₁₂ at amultiplicity of infection (MOI) of about 2 to about 5 particle formingunits (pfu) per cell to produce recombinant cell Trichoplusiani-pVL-nhFc_(ε)Rα₆₁₂. The infection was allowed to proceed at acontrolled temperature of 27° C. for 48 hours, to produce recombinantprotein PhFc_(ε)Rα₁₇₂. Following infection, cells were separated fromthe medium by centrifugation, and the medium was frozen at −70° C.

PhFc_(ε)Rα₁₇₂ was purified from the culture medium described immediatelyabove by affinity chromatography using an IgE antibody produced by themyeloma cell line U266DI (American Tissue Type Catalogue No. TIB196)linked to sepharose 4B. The amino acid composition and N-terminal aminoacid sequence of the affinity purified PhFc_(ε)Rα₁₇₂ were determinedusing methods standard in the art. The results indicated thatPhFc_(ε)Rα₁₇₂ was properly processed by the Trichoplusia ni cells.

Example 3

This example describes the biotinylation of a recombinant human Fc_(ε)Ralpha chain protein.

Affinity purified recombinant protein PhFc_(ε)Rα₁₇₂, prepared asdescribed above in Example 2, was biotinylated as follows. About 440micrograms (μg) of PhFc_(ε)Rα₁₇₂ were diluted in about 1.5 milliliter(ml) of acetate buffer (0.1 M NaAc, pH 5.5) containing about 200microliter (μl) of 0.1 M NaIO₄. The mixture was incubated for about 20minutes, on ice, and about 2 μl of glycerol was added following theincubation. The mixture was then dialyzed against about 2 liters ofacetate buffer in a 3 ml Slide-A-Lyzer cassette (available from Pierce,Rockford, Ill.), 2 times for about 2 hours each time. About 3.72 μg ofbiotin-LC-hydrazide (available from Pierce) was dissolved in about 200μl of dimethylsulfoxide (DMSO) and injected into the cassette. Thecassette was then rocked at room temperature for about 2 hours.Following the incubation, the mixture containing recombinant protein andbiotin dialyzed 3 times, a first time for about 18 hours and two timesfor about 2 hours, each time at 5° C. against phosphate buffered saline.The biotinylated protein was recovered from the dialysis, and isreferred to herein as PhFc_(ε)Rα₁₇₂-BIOT.

Example 4

This example describes detection of canine IgE in a solid-phase ELISAusing PhFc_(ε)Rα₁₇₂-BIOT.

Wells of two Immulon II microtiter plates (available from Dynatech,Alexandria, Va.) were coated with duplicate samples of about 100 μl/wellof various concentrations of purified canine IgE as denoted in FIG. 1.The canine IgE was obtained from a canine IgE producing hybridoma, suchas heterohybridoma 2.39 (described in Gebhard et al., Immunology85:429-434, 1995) and was diluted in a CBC buffer (15 mM Na₂CO₃ and 34.8mM NaHCO₃, pH 9.6. The coated plates were incubated overnight at 4° C.Following incubation, the canine IgE-containing solution was removedfrom each plate, and the plates were blotted dry. The plates were thenblocked using about 200 μl/well of 0.25% bovine serum albumin (BSA)contained in phosphate buffered saline (PBSB) for about 1 hour at roomtemperature. The plates were then washed four times with 0.05% Tween-20in PBS (PBST) using an automatic washer (available from Dynatech).Experimental samples consisting of about 100 μl/well of a 1:4000dilution of 40 μg/ml PhFc_(ε)Rα₁₇₂-BIOT (about 145 μg/ml; described inExample 3), contained in PBSB with 0.05%Tween-20 (PESBT) were added toeach well of one plate coated with canine IgE.

Control samples consisting of about 100 μl of biotinylated anti-canineIgE monoclonal antibody D9 (supplied by Dr. DeBoer, U. of Wisconsin,Madison, Wis.) were added to each well of the other plate coated withcanine IgE. The plates were incubated for 1 hour at room temperature andthen washed four times with PBST. About 100 μl of about 0.25 μg/mlstreptavidin conjugated to horseradish peroxidase (available fromKirkegaard and Perry Laboratories (KPL), Gaithersburg, Md.; diluted inPBST) was added to each well that received experimental or controlsamples. The plates were then incubated for 1 hour at room temperatureand washed four times with PBST. About 100 μl of TMB substrate(available from available from KPL), that had been pre-warmed to roomtemperature, was added. Plates were then incubated for 10 minutes atroom temperature and then about 100 μl/well of Stop Solution (availablefrom KPL) was added. Optical densities of wells were read on aSpectramax Microtiter Plate (available from Molecular Devices Inc.)reader at 450 nm within 10 minutes of adding the stop solution.

The results shown in FIG. 1 indicate that the alpha chain of humanFc_(ε)R detects the presence of canine IgE (closed squares) in asolid-phase assay in a similar manner as the control antibody that bindsspecifically to canine IgE (D9; closed circles).

Example 5

This example describes detection of plant allergen-specific canine IgEusing PhFc_(ε)Rα₁₇₂-BIOT.

Multiple wells of an Immulon II microtiter plate (available fromDynatech) were coated with either about 100 μl/well of 1 μg/ml ofKentucky Blue Grass allergen or about 100 μl/well of about 1 μg/ml ofGreen Ash allergen (both available from Greer Inc., Lenoir, N.C.) bothdiluted in CBC buffer. The plate was incubated overnight at 4° C. Theplate was blocked and washed as described in Example 4. Two differentpools of canine sera were then added to the antigen-coated wells. Thefirst pool consisted of sera isolated from 8 dogs reported to beallergen reactive. The second pool consisted of sera isolated from 8dogs reported to be allergen non-reactive. Each pool of sera was diluted1:10 or 1:100 in PBST. About 100 μl of each concentration of eachdiluted sera sample was added to the wells and incubated for 1 hour atroom temperature. The plate was then washed four times with PBST. About100 μl/well of a 1:4000 dilution of 40 g/ml PhFc_(ε)Rα₁₇₂-BIOT(described in Example 3), contained in PBSBT was added to theantigen-coated wells. The plate was incubated for 1 hour at roomtemperature. The plate was then washed four times with PBST. About 100μl/well of about 0.25 μg/ml of neutravidin conjugated to horseradishperoxidase (available from Pierce) contained in PBSBT, was added. Theplate was incubated for 1 hour at room temperature. The plate was thenwashed and the presence of neutravidin bound to the plate detected usingthe method described in Example 4.

The results shown in FIG. 2 indicate that the alpha chain of humanFc_(ε)R detects the presence of canine IgE antibodies that bindspecifically to a common grass allergen or to a common tree allergen. Inaddition, detection of canine IgE antibodies is dose dependent.

Example 6

This example describes detection of total canine IgE usingPhFc_(ε)Rα₁₇₂-BIOT.

Multiple wells of an Immulon II microtiter plate (available fromDynatech) were coated with about 100 μl/well of about 1 μg/ml CMIanti-canine IgE antibody #6 (available from Custom MonoclonalsInternational, West Scramento, Calif.) diluted in CBC buffer. The platewas incubated overnight at 4° C. The plate was blocked and washed asdescribed in Example 4. About 100 μl/well of a 1:60 dilution in PBSBT ofsera samples from a variety of sources were then added to multiple wellscoated with anti-IgE antibody. The samples included:(1) serum from a dogknown to be allergic to flea saliva; (2) serum from dogs infected withD. immitis; (3) and (4) a pool of dog sera from defined as canineallergy calibrators (available from BioProducts DVM, Tempe, Ariz.); (5)pools of dog sera containing antibodies that have low binding toKentucky Blue Grass allergen; (6) pools of dog sera that have highbinding to Kentucky Blue Grass allergen; (7) a pool of dog sera fromdogs known to be allergic to flea saliva, the sample was heatinactivated (at 56° C. for 4 hours); (8) a pool of dog sera from dogsknown to be allergic to flea saliva; or (9) a pool of dog sera from dogsraised in a barrier facility (i.e., negative control). A set of positivecontrol samples consisting of IgE derived from the canineheterohybridoma described in Example 4 were also added to the plate togenerate a standard curve. The plate was incubated for 1 hour at roomtemperature and then washed four times with PBST. The presence of canineIgE was detected using either about 100 μl/well of a 1:4000 dilution of40 μg/ml PhFc_(ε)Rα₁₇₂-BIOT (described in Example 3) or about 100μl/well of about 1 μg/ml CMI anti-canine IgE antibody #19 (availablefrom Custom Monoclonals International), both contained in PBSBT. Theplate was incubated for 1 hour at room temperature. The plate was thenwashed, contacted with about 0.25 μg/ml streptavidin conjugated tohorseradish peroxidase, washed again, and the presence of streptavidinbound to the plate was detected using the method described in Example 4.The optical density readings obtained for the control samples were usedto generate a standard curve that was used to determine the total IgEbound to wells that had received test samples.

The results shown in FIG. 3 indicate that canine IgE from a variety ofdog sera are detected using the alpha chain of human Fc_(ε)R in a mannersimilar to using an antibody that binds specifically to canine IgE. Theabsence of detectable amounts of IgE in the heat treated sample (Sample7) indicates that the antibody detected by PhFc_(ε)Rα₁₇₂-BIOT is IgE. Inaddition, the results indicate that PhFc_(ε)Rα₁₇₂-BIOT is an effectivereagent for detecting IgE that binds to allergen Kentucky Blue Grass,Samples 5 and 6), as well as a parasite antigen (D. Immitis, Sample 2).

Example 7

This example describes detection of canine IgE in dog sera isolated fromdogs known to be allergic to flea saliva, using PhFc_(ε)Rα₁₇₂-BIOT.

Multiple wells of an Immulon IT microtiter plate were coated with about100 μl/well of varying concentrations of flea saliva recombinant proteinfspN (described in PCT Patent Publication No. WO 96/11271, ibid.;concentrations shown in FIG. 4) diluted in CBC buffer. The plate wasincubated overnight at 4° C. The plate was then blocked and washed asdescribed in Example 4. About 100 μl/well of a 1:10 dilution in PBSBT ofa pool of sera isolated from dogs known to produce IgE that bindsspecifically to flea saliva. Some wells did not receive dog sera so thatbackground binding levels could be determined. The plate was incubatedfor 1 hour at room temperature and then washed four times with PBST.About 100 μl/well of a 1:4000 dilution of 40 μg/ml PhFc_(ε)Rα₁₇₂-BIOT(described in Example 3) contained in PBSBT was added. The plate wasincubated for 1 hour at room temperature. The plate was then washed,contacted with about 0.25 ug/ml streptavidin-conjugated to horseradishperoxidase, washed again, and the presence of streptavidin bound to theplate was detected using the method described in Example 4.

The results shown in FIG. 4 indicate that canine IgE that bindsspecifically to a flea saliva antigen is detected using the alpha chainof human Fc_(ε)R.

Example 8

This example describes detection of total canine IgE in dog seraisolated from dogs known to be allergic to flea saliva,heartworm-infected dogs and specific pathogen free (SPF) dogs, usingPhFc_(ε)Rα₁₇₂-BIOT.

Multiple wells of an Immulon II microtiter plate were coated with about100 μl/well of about 1 μg/ml CMI anti-canine IgE antibody #6 (availablefrom Custom Monoclonals International) in CBC buffer. The plate wasincubated overnight at 4° C. The plate was blocked and washed asdescribed in Example 4. About 100 μl/well of different samples ofIgE-containing fluids in PBSBT were added to multiple wells coated withthe anti-canine IgE antibody. The samples included: (1) 100 μg/ml ofcanine IgE purified from the heterohybridoma described in Example 4; (2)a 1:10 dilution of a pool of sera from dogs known to be allergic to fleasaliva, (3) a 1:10 dilution of the same sera pool as in (2) but heatinactivated; (4) a 1:10 dilution of serum from a dog known to haveclinical flea allergy dermatitis (dog CPO2); (5) a 1:10 dilution of heatinactivated CPO2 serum; (6) a 1:10 dilution of serum from aheartworm-infected dog (dog 417); (7) a 1:10 dilution of heatinactivated 417 serum; (8) a 1:10 dilution of a pool of sera fromheartworm-infected dogs; (9) a 1:10 dilution of the same sera pool as in(8) but heat inactivated; and (10) a pool of sera from dogs raised in abarrier facility. Each sample was diluted in PBSBT. The plate wasincubated for 1 hour at room temperature and then washed four times withPBST. About 100 μl/well of a 1:4000 dilution of 40 μg/mlPhFc_(ε)Rα₁₇₂-BIOT (described in Example 3) in PBSBT was added. Theplate was incubated for 1 hour at room temperature. The plate was thenwashed, contacted with about 0.25 ug/ml streptavidin-conjugated tohorseradish peroxidase, washed again, and the presence of streptavidinbound to the plate was detected using the method described in Example 4.

The results shown in FIG. 5 indicate that canine IgE from dogs allergicto flea saliva and from dogs infected with heartworm are detected usingthe alpha chain of human Fc_(ε)R. In addition, the absence ofcolorimetric signal in samples of heat inactivated sera indicates thatantibody bound to the anti-IgE antibody and detected by Fc_(ε)R alphachain is an epsilon isotype antibody and not another isotype.

Example 9

This example describes detection of IgE that specifically binds to fleasaliva, using PhFc_(ε)Rα₁₇₂-BIOT.

Multiple wells of an Immulon II microtiter plate were coated with about100 μl/well of about 0.1 μg/ml of flea saliva collected using the methoddescribed in PCT Patent Publication No. WO 96/11271, ibid., in CBCbuffer. The plate was incubated, blocked and washed as described inExample 4. The IgE-containing samples described in Example 8 were thenapplied to the flea saliva coated plate. The plate was then treatedusing the method described in Example 8.

The results shown in FIG. 6 indicate that canine IgE that bindsspecifically to flea saliva, contained in serum, is detected using thealpha chain of human Fc_(ε)R. In addition, the absence of calorimetricsignal in samples of heat inactivated serum indicates that antibodybound to the flea saliva protein and detected by Fc_(ε)R alpha chain isan epsilon isotype antibody.

Example 10

This example describes the detection of feline IgE usingPhFc_(ε)Rα₁₇₂-BIOT.

Multiple wells of an Immulon II microtiter plate were coated with about100 μl/well of about 10 μg/ml Di33 protein (described in U.S. patentapplication Ser. No. 08/715,628, ibid.) or 10 μg/ml crude homogenate ofheartworm, both in CBC buffer. Crude homogenate of heartworm is theclarafied supernatant of adult heartworms homogenized in PBS. The platewas incubated overnight at 4° C. The plate was blocked and washed asdescribed in Example 4. Serum samples from 2 heartworm infected catswere then added to Di33-coated wells and to heartworm antigen-coatedwells. About 100 μl/well of a 1:10 dilution in PBSBT of sera fromheartworm-infected cat # AXH3 or from cat #MGC2 were added to the plate.Negative control samples consisting of serum from pre-infection bleedsof cat #AXH3 and cat# MGC2 were also added to the plate at a dilution of1:10 in PBSBT. A positive control sample consisting of a pool of serafrom heartworm-infected dogs was also added to the plate at a dilutionof 1:10 in PBSBT. The plate was incubated for 1 hour at room temperatureand then washed four times with PBST. About 100 μl/well of a 1:4000dilution of 40 μg/ml PhFc_(ε)Rα₁₇₂-BIOT (described in Example 3) inPBSBT was added. The plate was incubated for 1 hour at room temperature.The plate was then washed, contacted with 1:4000 dilution of a 0.5 mg/mlsolution of streptavidin-conjugated to horseradish peroxidase, washedagain, and the presence of streptavidin bound to the plate was detectedusing the method described in Example 4.

The results shown in FIG. 7 indicate that feline IgE that bindsspecifically to crude homogenate of heartworm or Di33 protein isdetected using the alpha chain of human Fc_(ε)R.

Example 11

This example describes detection of feline IgE using PhFc_(ε)Rα₁₇₂-BIOT.Multiple wells of an Immulon II microtiter plate were coated with Di33as described in Example 10, in CBC buffer. The plate was incubatedovernight at 4° C. The plate was blocked and washed as described inExample 4. Serum samples from 2 heartworm infected cats were then addedto Di33-coated wells. About 100 μl/well of a 1:10 dilution in PBSBT ofserum from heartworm-infected cat # MGC2 and a pool of sera fromheartworm-infected cats, as well as heat inactivated samples of each ofthese sera, were added to the plate. A positive control sampleconsisting of a pool of sera from heartworm-infected dogs was also addedto the plate at a dilution of 1:10 in PBSBT. The plate was incubated for1 hour at room temperature and then washed four times with PBST. About100 μl/well of a 1:4000 dilution of 40 μg/ml PhFc_(ε)Rα₁₇₂-BIOT(described in Example 3) in PBSBT was added. The plate was incubated for1 hour at room temperature. The plate was then washed, contacted withstreptavidin-conjugated to horseradish peroxidase, washed again, and thepresence of streptavidin bound to the plate was detected using themethod described in Example 4.

The results shown in FIG. 8 indicate that feline IgE fromheartworm-infected cats that specifically binds to the heartworm antigenDi33 is detected using the alpha chain of human Fc_(ε)R. In addition,the absence of calorimetric signal in samples of heat inactivated seraindicates that antibody bound to the Di33 protein and detected byFc_(ε)R alpha chain is an epsilon isotype antibody.

Example 12

This example describes detection of equine IgE in a solid-phase ELISAusing PhFc_(ε)Rα₁₇₂-BIOT.

Horse sera from a horse known to be allergic to certain allergens andhorse sera from a horse known not to be allergic the same allergens,were assayed for the presence of IgE using PhFc_(ε)Rα₁₇₂-BIOT asfollows. A North Atlantic/Ohio Valley Regional Panel plate of a Canitec™Allergen-Specific IgE Kit (available from BioProducts DVM) was blockedand washed as described in Example 4. Two samples of about 1:10dilutions of the two horse sera were prepared using PBSBT. The twosamples were added to the blocked plate and the plate was incubated for1 hour at room temperature. The plate was washed as described in Example4. About 100 μl/well of a 1:4000 dilution of 40 μg/ml PhFc_(ε)Rα₁₇₂-BIOT(described in Example 3), contained in PBSBT was added to each well. Theplate was then washed, contacted with 1:4000 dilution of a 0.5 mg/mlsolution of streptavidin-conjugated to horseradish peroxidase, washedagain, and the presence of streptavidin bound to the plate was detectedusing the method described in Example 4.

The results shown in FIG. 9 indicate that equine IgE from a horse knownto be allergic to certain allergens specifically binds to certain plantand mite allergens is detected using the alpha chain of human Fc_(ε)R.

Example 13

This example describes detection of canine IgE in a solid-phase ELISAusing basophilic cells transfected with human Fc_(ε)R alpha chain.

Rat basophilic leukemia (RBL) cells transfected with a nucleic acidmolecule encoding a human Fc_(ε)R alpha chain (referred to herein asRBL-hFc_(ε)R cells; described in Miller et al., Science 244:334-337,1989) were used to detect canine IgE as follows. About 4×10⁴RBL-hFc_(ε)R cells contained in Earles Modified Eagles Medium containing10% fetal bovine serum (EMEM-FBS) were added to each well of 96-wellflat bottom tissue culture plates. The RBL-hFc_(ε)R cells were incubatedovernight at 37° C. Following the incubation the plates were washed 4times with PBST. The cells were then fixed for about 2 minutes usingabout 200 μl per well of absolute alcohol at room temperature. Theplates were then washed 8 times with PBST to remove residual alcohol.

Serial dilutions in EMEM-FBS (concentrations shown in FIG. 10) wereprepared using a pool of sera from dogs infected with heartworm. Serialdilutions in EMEM-FBS (concentrations shown in FIG. 11) were preparedusing a pool of sera from dogs sensitized to flea saliva. Additionalsamples were prepared in which both pools of sera were heat inactivatedfor about 4 hours at 56° C. The heat treated samples were diluted asdescribed above.

About 100 μl of each dilution of each serum sample was added to separatewells containing fixed RBL-hFc_(ε)R cells and the plates were incubatedat 37° C. for about 1 hour. Following the incubation, the plates werewashed 4 times with PBST. About 5 μg of a murine IgG monoclonal antibodyanit-canine IgE antibody (i.e., Custom Monoclonal Antibody #71;available from Custom Monoclonal International) in 100 μl of EMEM-FBSwas added to each well. The plates were incubated for about 30 minutesat 37° C. Following the incubation, the plates were washed 4 times withPBST. About 100 ng of horseradish peroxidase labelled donkey anti-murineIgG (available from Jackson Laboratories, Westgrove, Pa.) in 100 μlEMEM-FBS was added to each well, and the plates were incubated for about30 minutes at room temperature. Following the incubation, the plateswere washed 4 times with PBST. The presence of anti-murine IgG bound tothe plates thereby indicating the ability of RBL-hFc_(ε)R cells to bindto canine IgE was detected using the method described in Example 4.

The results shown in FIG. 10 indicate that canine IgE fromheartworm-infected dogs (♦) is detected using RBL-h Fc_(ε)R cellsexpressing the alpha chain of human Fc_(ε)R. In addition, the absence ofcolorimetric signal in samples of heat inactivated samples of such sera(▪) indicates that antibody detected by the Fc_(ε)R alpha chain on theRBL-h Fc_(ε)R cells is an epsilon isotype antibody. Similarly, theresults shown in FIG. 11 indicate that canine IgE from dogs sensitizedwith flea saliva (♦) is detected using RBL-h Fc_(ε)R cells expressingthe alpha chain of human Fc_(ε)R. In addition, the absence ofcolorimetric signal in samples of heat inactivated samples of such sera(▪) indicates that antibody detected by the Fc_(ε)R alpha chain on theRBL-h Fc_(ε)R cells is an epsilon isotype antibody.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

1. A method to detect a plant allergy in an animal comprising: (a) contacting a plant allergen with a putative IgE containing composition from said animal, under conditions suitable for formation of an antigen:IgE complex; (b) detecting the presence of a antigen:IgE complex, if present, by contacting the putative antigen:IgE complex with a FcεR molecule under conditions suitable for formation of an antigen:IgE:FcεR molecule complex, wherein the presence of an antigen:IgE:FcεR complex indicates the animal is allergic to the plant from which the allergen is derived.
 2. The method of claim 1, wherein said animal is a canid or a feuid.
 3. The method of claim 1, wherein said putative IgE containing composition comprises an animal fluid is selected from the group consisting of serum, plasma, blood, urine, tears, saliva, nasal secretions and milk.
 4. The method of claim 1, wherein said allergen is selected from the group consisting of a grass allergen, a plantain allergen, a pigweed allergen, a ragweed allergen, a sage allergen, an elm allergen, a cocklebur allergen, an elder allergen, a walnut allergen, a cottonwood allergen, an ash allergen, a birch allergen, a cedar allergen, an oak allergen, and a mulberry allergen.
 5. The method of claim 1, wherein said allergen is selected from the group consisting of a Johnson Grass allergen, a Kentucky Blue Grass allergen, a Meadow Fescue allergen, an Orchard Grass allergen, a Perennial Rye Grass allergen, a Redtop Grass allergen, a Timothy Grass allergen, a Bermuda Grass allergen, a Brome Grass allergen, a Curly Dock allergen, an English Plantain allergen, a Mexican Firebush allergen, a Lamb's Quarters allergen, a Rough Pigweed allergen, a Short Ragweed allergen, a Wormwood Sage allergen, an American Elm allergen, a Common Cocklebur allergen, a Box Elder allergen, a Black Walnut allergen, an Eastern Cottonwood allergen, a Green Ash allergen, a River Birch allergen, a Red Cedar allergen, a Red Oak allergen, and a Red Mulberry allergen.
 6. The method of claim 1, wherein said FcεR molecule is conjugated to a detectable marker.
 7. The method of claim 1, wherein said isolated FeεR molecule comprises at least a portion of a human FcεR alpha chain that binds to IgE.
 8. The method of claim 1, wherein said FcεR molecule comprises at least a portion of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:11 and SEQ ID NO:
 13. 9. The method of claim 1, wherein said FcεR molecule is encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ If) NO:7, SEQ ID NO:10 and SEQ ID NO:12.
 10. The method of claim l, wherein said allergen is bound to a substrate.
 11. A method to detect plant-allergen-specific IgE in an animal, said method comprising: (a) contacting a plant allergen with a putative IgE containing composition from said animal, under conditions suitable for formation of an antigen:IgE complex; (b) detecting the presence of said antigen:IgE complex, if present, by contacting the putative antigen:IgE complex with a FcεR molecule under conditions suitable for formation of an antigen:IgE:FcεR molecule complex, wherein the presence of an antigen:IgE:FcεR molecule complex indicates the presence of plant-allergen-specific IgE.
 12. The method of claim 11, wherein said animal is a canid or a felid.
 13. The method of claim 11, wherein said putative IgE containing composition comprises an animal fluid is selected from the group consisting of serum, plasma, blood, urine, tears, saliva, nasal secretions and milk.
 14. The method of claim 11, wherein said allergen is selected from the group consisting of a grass allergen, a plantain allergen, a pigweed allergen, a ragweed allergen, a sage allergen, an elm allergen, a cocklebur allergen, an elder allergen, a walnut allergen, a cottonwood allergen, an ash allergen, a birch allergen, a cedar allergen, an oak allergen, and a mulberry allergen.
 15. The method of claim 11, wherein said allergen is selected from the group consisting of a Johnson Grass allergen, a Kentucky Blue Grass allergen, a Meadow Fescue allergen, an Orchard Grass allergen, a Perennial Rye Grass allergen, a Redtop Grass allergen, a Timothy Grass allergen, a Bermuda Grass allergen, a Brome Grass allergen, a Curly Dock allergen, an English Plantain allergen, a Mexican Firebush allergen, a Lamb's Quarters allergen, a Rough Pigweed allergen, a Short Ragweed allergen, a Wormwood Sage allergen, an American Elm allergen, a Common Cocklebur allergen, a Box Elder allergen, a Black Walnut allergen, an Eastern Cottonwood allergen, a Green Ash allergen, a River Birch allergen, a Red Cedar allergen, a Red Oak allergen, and a Red Mulberry allergen.
 16. The method of claim 11, wherein said FcεR molecule is conjugated to a detectable marker.
 17. The method of claim 11, wherein said isolated FcεR molecule comprises at least a portion of a human FcεR alpha chain that binds to IgE.
 18. The method of claim 11, wherein said FcεR molecule comprises at least a portion of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:11 and SEQ ID NO:
 13. 19. The method of claim 11, wherein said FcεR molecule is encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10 and SEQ ID NO:12.
 20. The method of claim 11, wherein said allergen is bound to a substrate. 